Scios Inc.

Scios Nova, Inc.

Other

California Biotechnology Inc.

Biotechnology Research Partners, Ltd.

Eli Lilly and Company

Merrell Pharmaceuticals, Inc.

Byk Gulden Lomberg Chemische Fabrik Gmbh

Barbara Cordell

James W Schilling, Jr

Robert T White

Bradley J Benson

Yu-Wang Liu

Diana Hom Quon

Qiang Xu

Paul Andrew Hyslop

Jeffrey N Higaki

Ziyang Zhong

Sheila P Little

Edward Marion Johnstone

Frauke Schimmöller

Beverly Dale

Phyllis A Ponte

Steven Wyatt Queener

Tinggui Yin

Justin McCarthy

Norton P Peet

Linda Shawn Higgins

Mitchell Wayne Mutz

David Lawrence Wolf

Michael R Angelastro

Viviane Van Dorsselaer

Daniel G Schirlin

Steven Marc Paul

Kelly R Bales

Ronald P Mischak

Stewart A Thompson

Nobuhiko Katunuma

Guy Rautmann

Jan Marian Scardina

Edmund G Tischer

Rebecca Pruss

Frauke Schimm&ouml;ller

John Huggins

Foy Dean Miller

Rosanne Catalano

Elizbieta Puchacz

Asha Naidu

Frauke Schimmöoller

Linda Slanec Higgins

Daniel Schirlin

The present invention is based on the findings that BACE2, a homolog of &#x3b2;-secretase BACE, is able to stimulate processing of APP in a non-amyloidogenic pathway, thereby suppressing the level of A&#x3b2;. Accordingly, the present invention provides methods and means for the identification and use of modulators of this unique activity of BACE2 to suppress A&#x3b2; production. The compounds identified using the methods and means provided herein may be used as potential candidates for the treatment of Alzheimer's disease and other neurological diseases by reducing &#x3b2;-amyloid deposit formation.

Inhibition of A&#x3b2; production by &#x3b2;-secretase BACE2

The invention concerns a novel &#x3b2;-secretase, a method of partially purifying this novel &#x3b2;-secretase, and its use in assays to screen for potential drug candidates against Alzheimer's disease and other neurological diseases. The novel &#x3b2;-secretase has an estimated molecular weight of about 32-39 kDa or 22-26 kDa in HEK293 cell membrane extracts and human brain samples, respectively, as calculated from radiation inactivation analysis, and has a pH optimum at about pH 6.5-7.0.

&#x3b2;-secretase and modulation of &#x3b2;-secretase activity

The invention concerns a novel &#x3b2;-secretase, a method of partially purifying this novel &#x3b2;-secretase, and its use in assays to screen for potential drug candidates against Alzheimer's disease and other neurological diseases. The novel &#x3b2;-secretase has an estimated molecular weight of about 32&#x2013;39 kDa or 22&#x2013;26 kDa in HEK293 cell membrane extracts and human brain samples, respectively, as calculated from radiation inactivation analysis, and has a pH optimum at about pH 6.5&#x2013;7.0.

Disclosed herein are methods for identifying inhibitors of neuronal degeneration, and their use in the treatment of neurodegenerative disorders. In particular methods methods are disclosed for identifying inhibitors of neuronal degeneration or cell death by taking advantage of the involvement of presenilin (PS) and Par-4 in NF-&#x3ba;B activation.

Methods for identifying inhibitors of neuronal degeneration

The present invention concerns methods and means for identifying inhibitors of neuronal degeneration, and their use in the treatment of neurodegenerative disorders. In particular the invention concerns methods and means for identifying inhibitors of neuronal degeneration or cell death by taking advantage of the involvement of presenilin (PS) and Par-4 in NF-&#x3ba;B activation.

The present invention is based on the findings that BACE 2, a homolog of &bgr;-secretase BACE, is able to stimulate processing of APP in a non-amyloidogenic pathway, thereby suppressing the level of A&bgr;. Accordingly, the present invention provides methods and means for the identification and use of modulators of this unique activity of BACE 2 to suppress A&bgr; production. The compounds identified using the methods and means provided herein may be used as potential candidates for the treatment of Alzheimer's disease and other neurological diseases.

Modulation of A&bgr; levels by &bgr;-secretase BACE2

The invention relates to the design, construction and use of eukaryotic cell lines useful in the identification of inhibitors of &bgr;-amyloid processing. More specifically, the invention relates to in vitro assays capable of identifying or quantifying a 4.2 kDa &bgr;-amyloid protein. The present invention also provides for DNA and protein molecules for the design, construction and use of eukaryotic cell lines useful in the identification of inhibitors of &bgr;-amyloid processing.

Assays for &bgr;-amyloid processing

The present invention provides assays to identify compounds that affect microglial cell activation, and specifically assays to identify compounds that affect secretion of cytokines from these microglial cells by modulating PGE -mediated activity. The assays of the invention include assays for testing microglial cell activation by contacting microglia with compounds that modulate &bgr;-amyloid PGE -mediated activation, which can be identified by cellular activity such as secretion of cytokines, e.g., TNF-&agr; and IL-1&agr;. The effect of the candidate compound can be determined by comparing the effect with a control culture which is not contacted with the compound, or by comparing the effect with a standardized profile.

Prevention and treatment of amyloid-associated disorders

The present invention provides cell-free &ggr;-secretase activity. The method of the invention utilizes a membrane source of APP/&ggr;-secretase mixture in the assay to determine factors that may enhance or decrease enzymatic activity affecting &bgr;-amyloid peptide production. The cell membranes used in the assay may be from cells expressing an endogenous APP or, preferably, cells expressing a recombinant APP. The APP may be full-length or a fragment capable of being proteolytically cleaved by &ggr;-secretase. In addition, the APP expressed in the cells may have one or more mutation, such as a point mutation, small deletion, etc.

Modulation of &ggr;-secretase activity

The present invention provides a method of assaying for and arresting, preventing and/or reversing the impairment of central and peripheral nervous system function comprising reducing &bgr;-amyloid plaque burden by the administration of compounds that reduce apoE expression. The compounds used in the method of the invention may be: 1) inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase; 2) inhibitors of cholesterol biosynthesis; 3) inhibitors of protein isoprenylation, specifically geranylgeranylation; and/or 4) inhibitors of NF-&kgr;B activation or function. Assays for compounds with inhibit apoE expression from microglial cells are also disclosed.

Assay to identify compounds that alter apolipoprotein E expression

The present invention provides methods that can be used to identify &bgr;-amyloid reducing compounds. The present invention is based on the use of organotypic brain slice culturing methods to simultaneously assess the toxicity and &bgr;-amyloid reducing activity of a test compound.

Methods to identify &bgr;-amyloid reducing agents

Inhibitors of .beta.-amyloid protein production

Cloned recombinant or synthetic DNA sequences related to the pathology of Alzheimer's disease are injected into fertilized mammalian eggs (preferably mouse eggs). The injected eggs are implanted in pseudo pregnant females and are grown to term to provide transgenic mice whose cells express proteins related to the pathology of Alzheimer's disease. The injected sequences are constructed having promoter sequences connected so as to express the desired protein in specific tissues of the transgenic mammal (most notably in nerve tissue). The proteins which are preferably ubiquitously expressed include: (1) .beta.-amyloid core precursor proteins; (2) .beta.-amyloid related precursor proteins; and (3) serine protease inhibitor. The transgenic mice provide useful models for studying compounds being tested for their usefulness in treating Alzheimer's disease, and for studying the in vivo interrelationships of these proteins to each other.

Transgenic non-human mice displaying the amyloid-forming pathology of

Recombinant materials for the production of low molecular weight hydrophobic lung surfactant proteins are disclosed and claimed. The monomeric forms of the proteins from human and canine sources have apparent molecular weights of about 5 kd and form dimers under some conditions. The availability of these recombinant materials permits production of large amounts of these proteins through recombinant techniques and permits the use of these proteins in pharmaceutical compositions in the treatment of respiratory deficiency syndromes.

Recombinant alveolar surfactant protein

There are disclosed neurotrophic factors which are capable of being expressed as polypeptides lacking the microheterogeneity associated with a related native-sequence factor, CNTF. Also disclosed are DNA sequences encoding the neurotrophic factors and methods for expressing and recovering the factors as homogeneous polypeptides.

Production of homogeneous truncated CNTF

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Palo Alto, CA, United States of America

Barbara Cordell